Lung Fluid Clearance Research Articles

Clinical grade allogeneic human mesenchymal stem cells restore alveolar fluid clearance in human lungs rejected for transplantation

The lack of suitable donors for all solid-organ transplant programs is exacerbated in lung transplantation by the low utilization of potential donor lungs, due primarily to donor lung injury and dysfunction, including pulmonary edema. The current studies were designed to determine if intravenous clinical-grade human mesenchymal stem (stromal) cells (hMSCs) would be effective in restoring alveolar fluid clearance (AFC) in the human ex vivo lung perfusion model, using lungs that had been deemed unsuitable for transplantation and had been subjected to prolonged ischemic time. The human lungs were perfused with 5% albumin in a balanced electrolyte solution and oxygenated with continuous positive airway pressure. Baseline AFC was measured in the control lobe and if AFC was impaired (defined as <10%/h), the lungs received either hMSC (5 × 10(6) cells) added to the perfusate or perfusion only as a control. AFC was measured in a different lung lobe at 4 h. Intravenous hMSC restored AFC in the injured lungs to a normal level. In contrast, perfusion only did not increase AFC. This positive effect on AFC was reduced by intrabronchial administration of a neutralizing antibody to keratinocyte growth factor (KGF). Thus, intravenous allogeneic hMSCs are effective in restoring the capacity of the alveolar epithelium to remove alveolar fluid at a normal rate, suggesting that this therapy may be effective in enhancing the resolution of pulmonary edema in human lungs deemed clinically unsuitable for transplantation.

Acute Effects of Cigarette Smoke Extract on Alveolar Epithelial Sodium Channel Activity and Lung Fluid Clearance

Cigarette smoke contains high levels of reactive species. Moreover, cigarette smoke can induce cellular production of oxidants. The purpose of this study was to determine the effect of cigarette smoke extract (CSE)-derived oxidants on epithelial sodium channel (ENaC) activity in alveolar type 1 (T1) and type 2 (T2) cells and to measure corresponding rates of fluid clearance in mice receiving a tracheal instillation of CSE. Single-channel patch clamp analysis of T1 and T2 cells demonstrate that CSE exposure increases ENaC activity (NPo), measured as the product of the number of channels (N) and a channels open probability (Po), from 0.17 ± 0.07 to 0.34 ± 0.10 (n = 9; P = 0.04) in T1 cells. In T2 cells, CSE increased NPo from 0.08 ± 0.03 to 0.35 ± 0.10 (n = 9; P = 0.02). In both cell types, addition of tetramethylpiperidine and glutathione attenuated CSE-induced increases in ENaC NPo. Biotinylation and cycloheximide chase assays indicate that CSE-derived ROS increases channel activity, in part, by maintaining cell surface expression of the α-ENaC subunit. In vivo studies show that tracheal instillation of CSE promoted alveolar fluid clearance after 105 minutes compared with vehicle control (n = 10/group; P < 0.05).

The Fas/FasL pathway impairs the alveolar fluid clearance in mouse lungs

Alveolar epithelial damage is a critical event that leads to protein-rich edema in acute lung injury (ALI), but the mechanisms leading to epithelial damage are not completely understood. Cell death by necrosis and apoptosis occurs in alveolar epithelial cells in the lungs of patients with ALI. Fas activation induces apoptosis of alveolar epithelial cells, but its role in the formation of lung edema is unclear. The main goal of this study was to determine whether activation of the Fas/Fas ligand pathway in the lungs could alter the function of the lung epithelium, and the mechanisms involved. The results show that Fas activation alters the alveolar barrier integrity and impairs the ability of the lung alveolar epithelium to reabsorb fluid from the air spaces. This result was dependent on the presence of a normal Fas receptor and was not affected by inflammation induced by Fas activation. Alteration of the fluid transport properties of the alveolar epithelium was partially restored by β-adrenergic stimulation. Fas activation also caused apoptosis of alveolar endothelial cells, but this effect was less pronounced than the effect on the alveolar epithelium. Thus, activation of the Fas pathway impairs alveolar epithelial function in mouse lungs by mechanisms involving caspase-dependent apoptosis, suggesting that targeting apoptotic pathways could reduce the formation of lung edema in ALI.

Role of Alveolar β2-Adrenergic Receptors on Lung Fluid Clearance and Exercise Ventilation in Healthy Humans

BackgroundIn experimental conditions alveolar fluid clearance is controlled by alveolar β2-adrenergic receptors. We hypothesized that if this occurs in humans, then non-selective β-blockers should reduce the membrane diffusing capacity (DM), an index of lung interstitial fluid homeostasis. Moreover, we wondered whether this effect is potentiated by saline solution infusion, an intervention expected to cause interstitial lung edema. Since fluid retention within the lungs might trigger excessive ventilation during exercise, we also hypothesized that after the β2-blockade ventilation increased in excess to CO2 output and this was further enhanced by interstitial edema.Methods and Results22 healthy males took part in the study. On day 1, spirometry, lung diffusion for carbon monoxide (DLCO) including its subcomponents DM and capillary volume (VCap), and cardiopulmonary exercise test were performed. On day 2, these tests were repeated after rapid 25 ml/kg saline infusion. Then, in random order 11 subjects were assigned to oral treatment with Carvedilol (CARV) and 11 to Bisoprolol (BISOPR). When heart rate fell at least by 10 beats·min−1, the tests were repeated before (day 3) and after saline infusion (day 4). CARV but not BISOPR, decreased DM (−13±7%, p = 0.001) and increased VCap (+20±22%, p = 0.016) and VE/VCO2 slope (+12±8%, p<0.01). These changes further increased after saline: −18±13% for DM (p<0.01), +44±28% for VCap (p<0.001), and +20±10% for VE/VCO2 slope (p<0.001).ConclusionsThese findings support the hypothesis that in humans in vivo the β2-alveolar receptors contribute to control alveolar fluid clearance and that interstitial lung fluid may trigger exercise hyperventilation.

EtOH consumption contributes to a ‘pro‐injury’ environment in the alcoholic lung.

Chronic alcohol consumption increases ICU morbidity and mortality due to acute respiratory distress syndrome (ARDS). One reason is that alcohol regulates alveolar epithelial sodium channels (ENaC) via oxidant injury. We used mice to mimic acute and chronic alcohol consumption and tested whether ethanol (EtOH) would affect lung fluid clearance by altering lung ENaC activity. Chronic ethanol animals were tracheally instilled and imaged in vivo to observe lung fluid clearance. Lung fluid clearance showed a latent increase in clearance rates in mice receiving EtOH. Ethanol mice given an acute tracheal instillation of LPS and imaged for four hours showed lung fluid clearance early compared to control mice. But, ethanol mice with overnight exposure to LPS, showed reduced alveolar fluid clearance like the alveolar flooding seen in ARDS. Together, these data suggest that chronic EtOH consumption leads to altered regulation of ENaC, contributing to a ‘pro‐injury’ environment in the alcoholic lung. Funding support: R00HL09226 awarded to MNH; Children's Center for Development in Lung Biology Grant awarded to MNH.

Does Arhgef7, a guanine nucleotide exchange factor, regulate epithelial sodium channel activity and lung aeration?

Preterm infants have higher incidence of respiratory failure than term infants. These respiratory disorders are caused in part by delayed clearance of lung fluid. At birth, lung fluid clearance and aeration are mediated by active salt and water transport by epithelial sodium channel (ENaC) activity. Our lab has shown that oxidative environments can enhance ENaC activity, most likely via Nadph oxidase (Nox)‐mediated O2− release. In this highly regulated process, we have shown that the small G‐protein Rac1 plays an important role upstream of Nox signaling. However, the Rac1‐guanine nucleotide exchange factors (GEFs) involved in Nox‐mediated ENaC activity are unknown. Herein, we utilized quantitative real‐time PCR analysis to examine the expression profile of Rac1‐GEFs that may play an important role in ENaC regulation in the neonatal lung. Our major findings thus far are that transcript expression of Arhgef7&gt;;&gt;;&gt;;Sos1&gt;;Rasgrf2&gt;;Tiam1&gt;;Sos2, and that Arhgef7 transcript is expressed 1.5‐fold higher in neonatal (post‐partum day 7) mouse lung than adult mouse lung (n=8). This evidence suggests that Arhgef7 may play an integral role in neonatal lung aeration. Funding support: R00HL09226 awarded to MNH; Children's Center for Development in Lung Biology Grant awarded to MNH; and Emory University Summer Undergraduate Stipend awarded to DT.

Estradiol increases plasma membrane insertion of αENaC in the lung

Sodium reabsorption through alveolar epithelial sodium channels (ENaC) is the major driving force for lung fluid clearance. Recent evidence shows greater rates of lung fluid clearance in females than males suggesting that female sex hormones might regulate ENaC function. Using cell‐attached patch clamp, we have previously shown that β‐estradiol (E2) activates both highly‐selective and non‐selective ENaC in rat alveolar cells (L2). To extend that study, we examined possible mechanisms by which E2 increases ENaC activity. We first investigated whether E2 (200 pg/mL) altered αENaC gene expression in alveolar (L2) cells. E2 did not affect mRNA or protein levels of αENaC. Using cell surface biotinylation, we also examined differences in apical plasma membrane abundance of αENaC in vehicle and E2‐treated cells. There was ~3 fold increase in αENaC (60kDa) abundance with E2 treatment. Finally, we examined plasma membrane abundance of αENaC in lungs from female rats at the diestrus, proestrus, and estrus phases of the estrous cycle. Consistent with an effect of E2, we observed ~3 fold greater abundance of αENaC (60–75kDa) in lungs from proestrus rats. These results suggest that greater insertion of αENaC into the plasma membrane is one mechanism by which E2 increases alveolar ENaC activity. Future studies will focus on which estrogen receptor mediates this effect.Funding: NIH R37DK037963, K12GM000680 and T32HL076118

The association between cord hormones and transient tachypnea of newborn in late preterm and term neonates who were delivered by cesarean section

Objective: Failure of adequate and timely clearance of fetal lung fluid has been implicated in transient tachypnea of the newborn (TTN). There has been lack of human data on the association between endocrinological adaptation and fetal lung fluid clearance. Although TTN development in term or late preterm newborns delivered by cesarean section (CS) is well known, whether stress hormones levels at birth contribute to it or not is not known. The aim of the study was to assess the possible association between low adrenocorticothrophic hormone (ACTH), cortisol and free triiodothyronin (fT3) levels at birth and TTN in late preterm and term infants.Study design: We compared cord blood concentrations of epinephrine, cortisol, ACTH, fT4, fT3 and thyroid stimulating hormone in two groups of term and late pretrem infants born by CS: those who developed TTN and a comparison group without respiratory distress.Results: While there were no significant demographic differences between patient and control groups, cord ACTH, cortisol and fT3 were significantly lower and epinephrine was higher in infants developing TTN (p < 0.05).Conclusions: Lower cord levels of cortisol, ACTH and fT3 in patients with TTN may indicate the possible relation of these hormones in fetal lung fluid clearance and postnatal pulmonary adaptation through their modulatory effect on epithelial sodium channel and Na-K-ATPase.

Ethanol Alters Alveolar Fluid Balance via Nadph Oxidase (NOX) Signaling to Epithelial Sodium Channels (ENaC) in the Lung

Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS). However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC) via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH) would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a ‘pro-injury’ environment in the alcohol lung.

1806 Molecular Mechanisms of Perinatal Lung Fluid Clearance in term Newborns

Background and AimThe perinatal switch from secretion to absorption in airway fluid transport includes increase in gene expression and activity of ion channels, e.g. apical amiloride-sensitive epithelial sodium channel (ENaC)...

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

Hypoxia induced changes in lung fluid balance in humans is associated with beta-2 adrenergic receptor density on lymphocytes

BackgroundPrevious studies have demonstrated an important role for beta-2 adrenergic receptors (β2AR) in lung fluid clearance. The purpose of this investigation was to examine the relationship between β2AR density on lymphocytes and indices of lung water in healthy humans exposed to ∼17h of hypoxia (FIO2=12.5% in a hypoxia tent). MethodsThirteen adults (mean±SEM; age=31±3 years, BMI=24±1kg/m2, VO2 Peak=40±2 ml/kg/min) participated. Pulmonary function, CT derived lung tissue volume (Vtis-tissue, blood and water), lung diffusing capacity for carbon monoxide (DCO) and nitric oxide (DNO), alveolar-capillary conductance (DM), pulmonary capillary blood volume (Vc) and lung water (CT Vtis−Vc) were assessed before and after ∼17h normobaric hypoxia (FIO2=12.5%). β2AR density on lymphocytes was measured via radioligand binding. Arterial oxygen saturation (SaO2), cardiac output (Q), right ventricular systolic pressure (RVSP) and blood pressure (BP) were also assessed. ResultsAfter 17h hypoxia, SaO2 decreased from 97±1 (normoxia) to 82±4% and RVSP increased from 14±3 (normoxia) to 29±2mmHg (p<0.05) with little change in Q or BP. Vc and DM both increased with hypoxia with a small increase in DM/Vc ratio (p>0.05). CT Vtis decreased and lung water was estimated to decline 7±13%, respectively. β2AR density averaged 1497±187 receptors/lymphocyte and increased 21±34% with hypoxia (range −31 to +86%). The post-hypoxia increase in β2AR density was significantly related to the reduction in lung water (r=−0.64, p<0.05), with the subjects with the greatest increase in density demonstrating the largest decline in lung water. ConclusionsLung water decreases with 17h normobaric hypoxia are associated with changes in beta adrenergic receptor density on lymphocytes in healthy adults.

Epigenetic control of lung fluid clearance

Epigenetic control of lung fluid clearance

Transient Tachypnea of the Newborn: The Treatment Strategies

Transient tachypnea of the newborn results from delayed clearance of lung fluid and is a common cause of admission of full term and late preterm infants to neonatal intensive care units. The condition is particularly common after elective cesarean section. Conventional treatment involves supplemental oxygen, withholding enteral feeds and administration of intravenous fluids and antibiotics. Rarely, infants require CPAP and mechanical ventilation. Occasionally some babies develop severe hypoxemia and may require high concentrations of oxygen. The most effective strategy for accelerating reabsorbtion of fetal lung fluid is exogenous glucocorticoids. Potential therapies for TTN must be based on an understanding of the physiology of normal fetal lung fluid clearence at bith. Furosemide, racemic epinephrine and inhaled β-agonists have been studied for possible benefit in TTN. The routine administration of these drugs can not be recommended unless additional data become available.

Insulin up-regulates epithelial sodium channel in LPS-induced acute lung injury model in rats by SGK1 activation

Activity of the epithelial sodium channels (ENaCs) in the lung tissue plays a critical role on sodium/fluid homeostasis and the lung fluid clearance. The serum- and glucocorticoid-inducible kinase-1 (SGK1), one of the critical regulation proteins of ENaC, is activated by insulin and growth factors possibly through 3-phosphoinositide-dependent kinase PDK1 or/and phosphatidylinositol 3-kinase (PI3K). However, it is uncertain whether insulin shows its stimulatory action on ENaC by activation of SGK1 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) condition. In our study, Wistar rats were injected with LPS to induce ALI. Evans blue dye albumin (EBA) concentration was used to measure pulmonary oedema. For detecting the ratio of phospho-SGK1/SGK1 and α-ENaC protein, Western blot was performed. Real-time polymerase chain reaction (RT-PCR) was used to assess α-ENaC messenger RNA (mRNA). Immunohistochemistry was used to locate and quantitate α-ENaC expression. The EBA concentration was markedly increased by LPS alone but significantly reduced in rats that also received insulin injection. The ratio of phospho-SGK1/SGK1 was raised significantly in the insulin group and insulin+LPS group, compared with the control group and the LPS group, respectively. Furthermore, α-ENaC was up-regulated by insulin treatment. Simultaneously, injection with LPS significantly reduced α-ENaC expression. These findings demonstrated that insulin up-regulates ENaC in vivo possibly resulting from activation of SGK1.

β-Adrenergic agonists differentially regulate highly selective and nonselective epithelial sodium channels to promote alveolar fluid clearance in vivo

β-Adrenergic receptors (β-AR) increase epithelial sodium channel (ENaC) activity to promote lung fluid clearance. However, the effect of selective β-AR agonist on highly selective cation (HSC) channels or nonselective cation (NSC) channels in alveolar type 1 (T1) and type 2 (T2) cells is unknown. We hypothesized that stimulation with β(1)-AR agonist (denopamine) or β(2)-AR agonist (terbutaline) would increase HSC and/or NSC channel activity in alveolar epithelial cells. We performed single-channel measurements from T1 and T2 cells accessed from rat lung slices. Terbutaline (20 μM) increased HSC ENaC activity (open probability, NP(o)) in T1 (from 0.96 ± 0.61 to 1.25 ± 0.71, n = 5, P <0.05) and T2 cells (from 0.28 ± 0.14 to 1.0 ± 0.30, n = 8, P = 0.02). Denopamine (20 μM) increased NSC NP(o) in T1 cells (from 0.34 ± 0.09 to 0.63 ± 0.14, n = 7, P = 0.02) and in T2 cells (from 0.47 ± 0.09 to 0.68 ± 0.10, P = 0.004). In vivo X-ray imaging of lung fluid clearance and ICI 118,551 selective inhibition of β(2)-ARs confirmed patch-clamp findings. cAMP concentrations increased following treatment with denopamine or terbutaline (n = 3, P < 0.002). The effects of systemic (intraperitoneal, IP) and local (intratracheal, IT) modes of delivery on lung fluid clearance were assessed. IT delivery of denopamine promoted alveolar flooding, whereas IP delivery promoted delayed fluid clearance. In summary, β-AR agonists differentially regulate HSC and NSC in T1 and T2 cells to promote lung fluid clearance in vivo, and the mode of drug delivery is critical for maximizing β-AR agonist efficacy.

Chronic ethanol ingestion increases epithelial sodium channel activity in C57BL/6 lung

It is clear that a critical component of salt and water transport by the lungs depends on precise control of epithelial sodium channel (ENaC) activity, which generates the osmotic driving force for net transport of solute out of the air space. Chronic ethanol (EtOH) ingestion alters the signal transduction pathway leading to normal ENaC activity. We show that chronic EtOH ingestion in 12 week old C57BL/6 mice increases expression of ENaC, NADPH oxidase (Nox) 2, and Nox 4 protein (1.7, 1.5, and 2.8 fold, respectively) using western blot analysis. Real time PCR analysis of Rac‐guanine nucleotide exchange factors (which mediate activation of Nox) reveals that Arhgef7, Sos1, Sos2, Tiam1, and Ras‐GRF2 transcripts are significantly down‐regulated in EtOH lung compared to normal healthy lung. In vivo radiographic imaging indicates that these changes in cellular signaling lead to an enhanced rate of lung fluid clearance in EtOH animals following a tracheal saline challenge of 5uL/g body weight. We conclude that chronic ethanol ingestion activates NADPH oxidase, which oxidizes the alveolar environment in order to stimulate redox sensitive ENaC activity. This research was supported by SIRE awarded to DT, and R00‐HL‐09222601.

Epigenetic control of lung fluid clearance

The Na,K‐ATPase regulates ion transport and alveolar fluid clearance (AFC) in the lung. TGF‐β mediates acute lung injury (ALI), and impacts AFC. We hypothesized that TGF‐β impacts Na,K‐ATPase activity by modifying subunit stoichiometry. Cell surface density of the Na,K‐ATPase is regulated by the ATP1B1 subunit. ATP1B1 expression was reduced both in TGF‐β‐treated lung epithelial cells and in lungs from ALI patients or mice with experimental (bleomycin‐induced) lung injury. Luciferase, surface biotinylation and 86Rb+ uptake assays demonstrated that TGF‐β reduced Na,K‐ATPase gene expression, activity, and cell‐surface density, respectively, in primary lung epithelial cells. RNA interference and chromatin immunoprecipitation studies revealed that ATP1B1 downregulation relied on SMAD4, SNAI1 and E2F5 transcription factors, and was critically dependent on HDAC2. The HDAC inhibitor, Trichostatin A, improved edema resolution in experimental lung injury. Collectively, our data suggest that TGF‐β downregulates ATP1B1 expression leading to decreased Na,K‐ATPase activity and consequently impaired AFC in ALI, and that these processes can be reversed by HDAC inhibition.German Research Foundation

Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via O2− signaling

To define roles for reactive oxygen species (ROS) and epithelial sodium channel (ENaC) in maintaining lung fluid balance in vivo, we used two novel whole animal imaging approaches. Live X-ray fluoroscopy enabled quantification of air space fluid content of C57BL/6J mouse lungs challenged by intratracheal (IT) instillation of saline; results were confirmed by using conventional lung wet-to-dry weight ratios and Evans blue as measures of pulmonary edema. Visualization and quantification of ROS produced in lungs was performed in mice that had been administered a redox-sensitive dye, hydro-Cy7, by IT instillation. We found that inhibition of NADPH oxidase with a Rac-1 inhibitor, NSC23766, resulted in alveolar flooding, which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2(-/-) mice showed increased retention of air space fluid compared with wild-type controls. Interestingly, fluoroscopic analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo that was abrogated by the ROS scavenger tetramethylpiperidine-N-oxyl (TEMPO). Acute application of LPS increased the activity of 20 pS nonselective ENaC channels in rat type 1 cells; the average number of channel and single-channel open probability (NPo) increased from 0.14 ± 0.04 to 0.62 ± 0.23. Application of TEMPO to the same cell-attached recording caused an immediate significant decrease in ENaC NPo to 0.04 ± 0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity.

Regulation of alveolar fluid clearance and ENaC expression in lung by exogenous angiotensin II

Angiotensin II (Ang II) has been demonstrated as a pro-inflammatory effect in acute lung injury, but studies of the effect of Ang II on the formation of pulmonary edema and alveolar filling remains unclear. Therefore, in this study the regulation of alveolar fluid clearance (AFC) and the expression of epithelial sodium channel (ENaC) by exogenous Ang II was verified. SD rats were anesthetized and were given Ang II with increasing doses (1, 10 and 100 μg/kg per min) via osmotic minipumps, whereas control rats received only saline vehicle. AT1 receptor antagonist ZD7155 (10 mg/kg) and inhibitor of cAMP degeneration rolipram (1 mg/kg) were injected intraperitoneally 30 min before administration of Ang II. The lungs were isolated for measurement of alveolar fluid clearance. The mRNA and protein expression of ENaC were detected by RT-PCR and Western blot. Exposure to higher doses of Ang II reduced AFC in a dose-dependent manner and resulted in a non-coordinate regulation of α-ENaC vs. the regulation of β- and γ-ENaC, however Ang II type 1 (AT1) receptor antagonist ZD7155 prevented the Ang II-induced inhibition of fluid clearance and dysregulation of ENaC expression. In addition, exposure to inhibitor of cAMP degradation rolipram blunted the Ang II-induced inhibition of fluid clearance. These results indicate that through activation of AT(1) receptor, exogenous Ang II promotes pulmonary edema and alveolar filling by inhibition of alveolar fluid clearance via downregulation of cAMP level and dysregulation of ENaC expression.